Method for preparing glucagon-like peptides

Abstract

The present invention refers to a method for preparing a glucagon-like peptide, comprising precipitation of the peptide or of a precursor peptide by means of mixing with an anti-solvent comprising diisopropyl ether and acetonitrile. Further, the present invention also relates to a peptide conjugated to a solid phase and a pharmaceutical composition comprising a Liraglutide peptide obtainable from a method according to the present invention.

The invention claimed is: 
     
       1. A method for preparing a Liraglutide peptide or a salt thereof, comprising:
 (i) providing a solution S comprising a peptide of formula I: 
 
       
         
           
                 
               
                   His-Ala-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser- 
                 
                   Tyr-Leu-Glu-Gly-Gln-Ala-Ala-B 1 -Glu-Phe-Ile-Ala- 
                 
                   Trp-Leu-Val-Arg-Gly-Arg-Gly, 
                 
             
                
                
                
               
            
           
         
         wherein B 1  is Lys(palmitoyl-Glu-OH) or Lys(H-Glu-OH); 
         (ii) precipitation of the peptide of step (i) by mixing solution S with an anti-solvent comprising at least 50% (v/v) of diisopropyl ether and acetonitrile, wherein the volume ratio (diisopropyl ether:acetonitrile) is in the range of from (3:1) to (5:1); and 
         (iii) isolating the precipitate obtained from step (ii). 
       
     
     
       2. The method according to  claim 1 , wherein step (i) comprises:
 (i-a) providing a precursor peptide conjugated to a solid phase: 
 
       
         
           
                 
               
                   His-Ala-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser- 
                 
                   Tyr-Leu-Glu-Gly-Gln-Ala-Ala-B 2 -Glu-Phe-Ile-Ala- 
                 
                   Trp-Leu-Val-Arg-Gly-Arg-Gly-[resin], 
                 
             
                
                
                
               
            
           
         
         wherein B 2  is Lys (palmitoyl-Glu-OR 1 ) or B 2  is Lys (R 2 -Glu-OR 1 ), with R 1  being a carboxylic acid protecting group and R 2  being an amino protecting group; and wherein at least the side chains of Glu, Asp, and Lys bear protecting groups; and 
         (i-b) cleaving the precursor peptide off the resin. 
       
     
     
       3. The method according to  claim 2 , wherein step (i-a) comprises Fmoc-based Solid Phase Peptide Synthesis using suitably protected amino acid derivatives or dipeptide derivatives, wherein said protected amino acid derivatives or dipeptide derivatives are activated by one or more coupling reagent/additive mixtures selected for each step independently from the group consisting of:
 (A) (benzotriazolyl)tetramethyluronium tetrafluoroborate (TBTU)/diisopropylethylamine (DIPEA); 
 (B) diisopropylcarbodiimide (DIC)/cyano-hydroxyimino-acetic acid ethyl ester; 
 (C) 3-(diethoxy-phosphoryloxy)-3H-benzo[d][1,2,3]triazin-4-one (DEPBT)/DIPEA; and 
 (D) DIC/hydroxybenzotriazole (HOBt). 
 
     
     
       4. The method according to  claim 2 , wherein one or more pseudoproline dipeptides are introduced at a position selected from the group consisting of Gly 4 -Thr 5 , Phe 6 -Thr 7 , Thr 7 -Ser 8 , Val 10 -Ser 11 , and Ser 11 -Ser 12  of the peptide of formula I. 
     
     
       5. The method according to  claim 2 , wherein the N-terminal histidine moiety is introduced into the precursor peptide conjugated to the solid phase using an amino acid derivative selected from the group consisting of Boc-His(Boc)-OH, Boc-His(1-Trt)-OH, and Fmoc-His(1-Trt)-OH and the coupling reagent/additive mixture DEPBT/DIPEA. 
     
     
       6. The method according to  claim 3 , wherein the Fmoc protecting group is cleaved off the growing peptide chain conjugated to the solid phase using a mixture selected from the group consisting of 5-50% (v/v) piperidine or 4-methyl piperidine in N,N-dimethylformamide (DMF), 5-50% (v/v) piperidine or 4-methyl piperidine in N-methylpyrrolidone (NMP), 1-5% (v/v) diazabicyclo[5.4.0]undec-7-ene (DBU) in DMF, and 50% (v/v) morpholine in DMF. 
     
     
       7. The method according to  claim 1 , further comprising a step of reacting an activated ester of palmitic acid, with the Lys(H-Glu-OH) moiety of the peptide of formula I or with a Lys(H-Glu-OR 1 ) moiety obtainable by cleaving off the amino protecting group R 2  from the precursor peptide provided in step (i-a). 
     
     
       8. The method according to  claim 1 , wherein the solution S obtained from step (i) further comprises trifluoroacetic acid (TFA) and one or more scavengers. 
     
     
       9. The method according to  claim 1 , wherein the anti-solvent used in step (ii) comprises at least 75% (v/v), of a mixture M of diisopropyl ether and acetonitrile, and wherein the volume ratio (diisopropyl ether:acetonitrile) in said mixture M is in the range of from (3:1) to (5:1). 
     
     
       10. The method according to  claim 1 , wherein step (ii) comprises mixing the anti-solvent with solution S obtained from step (i) by:
 (ii-a) pre-mixing diisopropyl ether and acetonitrile before mixing it with the solution S obtained from step (i); or 
 (ii-b) first mixing diisopropyl ether with the solution S obtained from step (i) and subsequently mixing acetonitrile with the mixture comprising solution S and diisopropyl ether; or 
 (ii-c) first mixing acetonitrile with the solution S obtained from step (i) and subsequently mixing diisopropyl ether with the mixture comprising solution S and acetonitrile. 
 
     
     
       11. The method according to  claim 1 , wherein step (ii) is carried out at a temperature in the range of −5° C. to 10° C. 
     
     
       12. The method according to  claim 1 , wherein in step (iii), the precipitate obtained from step (ii) is isolated by filtration, centrifugation, or a combination of filtration and centrifugation.